Study on host adaptation and splicing regulation of NS segment of influenza A viruses

Abstract

Influenza A viruses can sometimes undergo cross-species transmission. Many host adaptation markers have been identified in the hemagglutinin (HA) and polymerase basic 2 (PB2) genes. Adaptive mutations in the HA mainly change the receptor binding specificity, while those in PB2 promote virus replication in host cells. Non-structural protein 1 (NS1) is an important virulence factor with multiple functions. Phylogenetic analysis suggests that the NS segment of influenza virus is associated with host range. However, no host adaptation marker has yet been characterized in the NS segment.

Through sequence analysis and laboratory passage of influenza virus strains, I identified two mutations, D101N (G327A) and E172K (G540A), in the NS segment of H7N9 virus. I demonstrated that D101N (G327A) improves general fitness of H7N9 virus in various cells and promotes H3N2 virus replication in human cells. My study found that the E172K (G540A) substitution first emerged in H9N2 virus, becoming predominant in this subtype since 2012, and was subsequently passed on to the H7N9 virus. The E172K (G540A) substitution specifically enhances replication efficiency of the 2013 H7N9 virus in human cells, while replicative ability in avian cells is retained.

Influenza virus utilizes host splicing machinery to process viral transcripts expressed from both M and NS segments. The splicing process is well controlled by many cis-regulatory elements and trans-regulatory factors. Functional characterization showed that the G540A substitution in NS mRNA is actually located within a previously undefined exonic splicing enhancer (ESE) motif. SF2/ASF, an SR protein, recognizes this novel ESE motif to enhance splicing efficiency of NS mRNA. My study further showed that back mutation to A540G (K172E) significantly enhances binding affinity of SF2/ASF to the ESE element, thereby increasing the splicing ratio of nuclear export protein (NEP) to NS1. Enhanced splicing efficiency leads to lower levels of NS1 protein expression and higher abundance of NEP protein during virus replication. This down regulation of NS1 expression results in a dramatic decrease in the expression of M1 protein, leading to attenuation of virus replication.

This study also found that the NS1 protein involved in regulation of NS mRNA splicing. I showed that the NS1 protein directly interacts with SF2/ASF in the nucleus and negatively modulates splicing of NS mRNAs during virus infection. NS1 protein controls the splicing of its own mRNA over the course of infection by shuttling between the nucleus and cytoplasm. Interestingly, the amino acid mutation K172E can significantly reduce the inhibitory effect of NS1 on splicing, indicating that influenza virus adopts multiple mechanisms to control NS mRNA splicing and balance the levels of NEP and NS1 expression.

In summary, this study provides an insight into the mechanisms involved in the regulation of NS mRNA splicing, both in cis and in trans. My findings demonstrate that avian influenza virus may utilize host adaptive strategies whereby NS mRNA splicing ratio is controlled to optimize replication efficiency and overcome host barriers.